Targeted alteration of dna with oligonucleotides

ABSTRACT

The current invention relates to a method for targeted alteration of acceptor DNA, for example duplex acceptor DNA. The method comprises use of at least two oligonucleotides, each oligonucleotide having at least one mismatch relative to the targeted (duplex) acceptor DNA. The mismatch of the first oligonucleotide is directed to a nucleotide at a position in the first strand of the duplex and the mismatch of the second oligonucleotide is directed to the nucleotide in the second strand that occupies the complementary position in the duplex acceptor DNA (e.g. forms a base-pair with the nucleotide in the first strand). These mismatches are located at specific positions within said oligonucleotides. Also provided is a kit that comprises instructions for performing the method according to the inventions, and in a preferred embodiment, comprises oligonucleotides suitable for use in the method.

TECHNICAL FIELD

The current invention relates to a method for targeted alteration ofacceptor DNA, for example duplex acceptor DNA. The method comprises useof at least two oligonucleotides, each oligonucleotide having at leastone mismatch relative to the targeted (duplex) acceptor DNA. Themismatch of the first oligonucleotide is directed to a nucleotide in thefirst strand of the duplex and the mismatch of the secondoligonucleotide is directed to the nucleotide in the second strand thatforms a base-pair with the nucleotide in the first strand. Thesemismatches are located at specific positions within saidoligonucleotides. Also provided is a kit that comprises instructions forperforming the method according to the inventions, and in a preferredembodiment, comprises oligonucleotides suitable for use in the method.

BACKGROUND OF THE INVENTION

Genetic modification is the process of deliberately creating changes inthe genetic material of living cells. Often the purpose is to modify agenetically encoded biological property of that cell, or of the organismof which the cell forms part or into which it can regenerate. Thesechanges can take the form of deletion of parts of the genetic material,addition of exogenous genetic material, or changes in the existingnucleotide sequence of the genetic material, for example by substitutingone nucleotide for another.

Methods for the genetic modification of eukaryotic organisms have beenknown for over 20 years, and have found widespread application in plant,human and animal cells and microorganisms for improvements in the fieldsof agriculture, human health, food quality and environmental protection.

A common genetic modification methodology consists of adding exogenousDNA fragments to the genome of a cell, which may then confer a newproperty to that cell or organism over and above the properties encodedby already existing genes (including applications in which theexpression of existing genes will thereby be suppressed).

Although these methods may have some effectiveness in providing thedesired properties to a target, these methods are nevertheless not veryprecise. There is, for example, no control over the genomic positions inwhich the exogenous DNA fragments are inserted (and hence over theultimate levels of expression). In addition, the desired effect willhave to manifest itself over the natural properties encoded by theoriginal and well-balanced genome. On the contrary, methods of geneticmodification that will result in the addition, deletion or conversion ofnucleotides in predefined genomic loci will allow the precise andcontrollable modification of existing genes.//esp

Oligonucleotide-directed Targeted Nucleotide Exchange (TNE) is a methodthat is based on the delivery into the eukaryotic cell of (synthetic)oligonucleotides (molecules consisting of short stretches of nucleotidesand/or nucleotide-like moieties that resemble DNA in their Watson-Crickbase pairing properties, but may be chemically different from DNA;(Alexeev and Yoon, 1998); (Rice et al., 2001); (Kmiec, 2003)).

By deliberately designing a mismatch nucleotide in the homology sequenceof the oligonucleotide, the mismatch nucleotide may induce changes inthe genomic DNA sequence to which the nucleotide may hybridize. Thismethod allows the conversion of one or more nucleotides in the target,and may, for example, be applied to create stop codons in existinggenes, resulting in a disruption of their function, or to create codonchanges, resulting in genes encoding proteins with altered amino acidcomposition (protein engineering).

Targeted nucleotide exchange (TNE) has been described in many organismsincluding plant, animal and yeast cells and is also referred to asOligonucleotide-directed Mutagenesis (ODM).

The first examples of TNE using chimeric DNA: RNA oligonucleotides camefrom animal cells (reviewed in (Igoucheva et al., 2001)). TNE usingchimeric DNA:RNA oligonucleotides has also been demonstrated in plantcells (Beetham et al., 1999; Kochevenko and Willmitzer, 2003; Okuzakiand Toriyama, 2004; Zhu et al., 2000; Zhu et al., 1999). In general, thefrequencies reported in both plant and animal studies were too low forpractical application of TNE on non-selectable chromosomal loci. TNEusing chimeric oligonucleotides was also found to be difficult toreproduce (Ruiter et al., 2003), resulting in a search for alternativeoligonucleotide designs giving more reliable results.

Several laboratories have focused on the use of single stranded (ss)oligonucleotides for TNE. These have been found to give morereproducible results in both plant and animal cells (Liu et al., 2002)(Parekh-Olmedo et al., 2005) (Dong et al., 2006). However, the greatestproblem facing the application of TNE in cells of, in particular, higherorganisms such as plants remains the relative low efficiency that hasbeen reported so far. In maize a conversion frequency of 1×10⁻⁴ has beenreported (Zhu et al., 2000). Subsequent studies in tobacco (Kochevenkoand Willmitzer, 2003) and rice (Okuzaki and Toriyama, 2004) havereported frequencies of 1×10⁻⁶ and 1×10⁻⁴ respectively.

TNE using various types of oligonucleotides has been the subject ofvarious patent and patent applications including U.S. Pat. No.6,936,467, U.S. Pat. No. 7,226,785, US579597, U.S. Pat. No. 6,136,601,US2003/0163849, US2003/0236208, WO03/013226, U.S. Pat. No. 5,594,121 andWO01/92512.

In U.S. Pat. No. 6,936,467 it is contemplated that the low efficiency ofgene alteration obtained using unmodified DNA oligonucleotides is theresult of degradation of the donor oligonucleotides by nucleases presentin the reaction mixture or the target cell. It is proposed toincorporate modified nucleotides that render the resultingoligonucleotides (more) resistant against nucleases. These modificationsare disclosed to preferably be located at the ends of theoligonucleotide whereas the mismatch is present at least 8 nucleotidesfrom each terminal end.

U.S. Pat. No. 7,226,785 also discloses methods for targeted chromosomalgenomic alterations using modified single-stranded oligonucleotides withat least one modified nuclease-resistant terminal region. TNE usingmodified single stranded oligonucleotides is also the subject ofWO02/26967.

Because of the low efficiency of the current methods of TNE thereremains a need for alternative and/or better TNE techniques. These canbe used alone or in combination with existing TNE techniques, like thosedisclosed above and in the art, to improve efficiency. Accordingly, thepresent inventors have set out to improve on the existing TNEtechnology.

SUMMARY OF THE INVENTION Technical Problem

The technical problem identified in the art is that the currentavailable methodology for introducing specific and desired geneticchanges in cells, for example for introducing specific genetic changesin the genome present in a plant cell, are hindered by low efficiency,making the techniques laborious and costly. There is a need to come toalternative and better TNE techniques.

One of the problems to be solved is therefore to provide for analternative and/or better and/or additional method for the introductionof genetic change(s) in the genetic information, in particular duplexDNA sequences, as is present in cells. Preferably such method hasimproved efficiency in comparison to those described in the art. Suchmethod would allow for the provision of cells with altered geneticinformation, more in particular for cells wherein a functionality of thecell has been changed by the introduction of the alteration in thetarget DNA. Such functionality may for example relate to alteredproperties of the protein encoded by a DNA sequence encompassing the DNAthat has been altered by the method according to the invention.

The Solution to the Problem

The solution to the problem is presented in the accompanying claims.

Duplex or double-stranded DNA is a term very well known to the skilledperson and refers to the two strands of DNA held in a double helix bycomplementary base pairing (Watson-Crick base-pairing) between A's andT's and between G's and C's.

The inventors have now found a new method for targeted alteration of aduplex DNA sequence comprising a first DNA sequence (comprised in thefirst strand) and a second DNA sequence (comprised in the second strand)which is the complement of the first DNA sequence.

The method takes advantage of at least two different and specificallydesigned donor oligonucleotides. Each of the two donor oligonucleotidescomprises a domain that is capable of hybridizing to the target (underconditions that allow hybridization, as they are known to the skilledperson). Each of the two donor nucleotides further comprises at leastone mismatch in comparison to the targeted duplex DNA sequence, whichmismatch is to be introduced in the targeted duplex DNA sequence.

The first oligonucleotide comprises a domain that is capable ofhybridizing to said first DNA sequence (in the first strand) and thesecond oligonucleotide comprises a domain that is capable of hybridizingto said second DNA sequence (in the second strand).

The at least one mismatch in the first oligonucleotide isdirected/relative to a nucleotide in the first DNA sequence and the atleast one mismatch in the second oligonucleotide is directed/relative tothe nucleotide in the second DNA sequence that forms a base-pair withthe nucleotide in the first DNA sequence in the duplex DNA.

In the art it is advocated and common knowledge that a mismatch in aoligonucleotide should be present within the oligonucleotide, in otherwords “somewhere in the middle” of the oligonucleotide (see for examplethe various patent application discussed above, in particular U.S. Pat.No. 6,936,467 and U.S. Pat. No. 7,226,785).

Such oligonucleotide-design from the art, with a mismatch somewhere inthe middle and flanked by various nucleotides at both sides, wouldprevent any skilled person from utilizing a set of at least twooligonucleotides as described above as these oligonucleotides will atleast partially share complementary domains that may for examplehybridize with each other therewith preventing use in targetednucleotide exchange.

However, it has, surprisingly and unexpectedly, been found that themethod according to the invention, using the at least twooligonucleotides described in detail herein, can be performed with goodefficiency when the mismatch in each of the oligonucleotide is notlocated somewhere in the middle of the oligonucleotide but at specificlocations. In particular it has been found that for efficient TNE themismatch in the at least two oligonucleotides described herein should(for each oligonucleotide independently) be located at most two,preferably at most one nucleotide from the 3′ end of an oligonucleotide.Most preferably the at least one mismatch is at the 3′ end of the (ss)oligonucleotide.

In contrast to the general belief that any mismatch should be in acentral part of a oligonucleotide, and that, for example, modificationsat the 5′ end and the 3′ end of the oligonucleotide should be introducedto prevent premature degradation of the oligonucleotide by nucleases(see e.g. U.S. Pat. No. 6,936,467), it was now found that having amismatch in the oligonucleotide zero, one or at most two nucleotide(s)from the 3′ end provides for oligonucleotides that can advantageously beused in methods of targeted nucleotide exchange, i.e. in methods fortargeted alteration of a duplex DNA sequence as described herein.

With the above it has now become possible to target at the same time anucleotide in the first DNA sequence and the nucleotide in the secondDNA sequence that forms a base-pair with the nucleotide in the first DNAsequence in the duplex DNA by using the at least two oligonucleotides asdescribed herein, further unexpectedly improving targeted nucleotideexchange.

Each of the oligonucleotides comprising the at least one mismatch zero,one or at most two nucleotide(s) from the 3′ end and as described hereinmay be further modified by the inclusion of modified nucleotides, i.e.nucleotides having a base modification, a backbone modification, a sugarmodification and/or a modification at the 3′ end and/or 5′ end of saidnucleotide. These modifications include well-known modifications toeither improve binding/hybridization of the oligonucleotides to thetarget sequence and/or to prevent or inhibit breakdown of theoligonucleotides by so-called nucleases. Examples of such modifiednucleotides include locked nucleic acids, or nucleotides havingphosphorothioate linkages. However, as shown in example 2, it is notrequired that the first or the second oligonucleotide according to theinvention incorporates nucleotides having phosphorothioate linkages noris it required that any other type of modified nucleotide isincorporated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Nucleotide sequence of the GFP ORF containing a stop codon (SEQID NO:1).

FIG. 2 Amino acid sequence of the GFP-STOP protein. The position of thestop codon is represented by the asterisk (SEQ ID NO:2).

FIG. 3 The constructs used in this study.

FIG. 4 Data showing TNE efficiency with oligonucleotides according tothe invention.

FIG. 5 shows the nucleotide sequence of the YFP-STOP construct (SEQ IDNO:12). The nucleotide at position 186 has been altered (C to A),resulting in an in-frame stop codon.

FIG. 6 shows the protein sequence of the YFP-STOP (SEQ ID NO:13). Theposition of the stop codon in the protein is indicated by an asterisk.

DEFINITIONS

In the following description and examples, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided. Unless otherwise defined herein,all technical and scientific terms used have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. The disclosures of all publications, patentapplications, patents and other references are incorporated herein intheir entirety by reference.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. For example, amethod for isolating “a” DNA molecule, as used above, includes isolatinga plurality of molecules (e.g. 10's, 100's, 1000's, 10's of thousands,100's of thousands, millions, or more molecules). In particular, theinvention described herein takes advantage of the use of at least twooligonucleotides. Where in the description reference is made to “a” or“the” oligonucleotide, this is not to be understood by the skilledperson to indicate the absence of one of the at least twooligonucleotides, but is to be understood to indicate that reference ismade, independently, to one, two, or more or all of the at least twooligonucleotides applied in the method according to the invention,unless the context clearly dictates otherwise. For example, if it ismentioned that the oligonucleotide may comprise an LNA-nucleotide, thisis to be understood by the skilled person that one of theoligonucleotides may comprise such LNA-residue, but also that both ofthe at least two oligonucleotides may comprise such LNA-residue.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded.

Methods of carrying out the conventional techniques used in method ofthe invention will be evident to the skilled worker. The practice ofconventional techniques in molecular biology, biochemistry,computational chemistry, cell culture, recombinant DNA, bioinformatics,genomics, sequencing and related fields are well-known to those of skillin the art and are discussed, for example, in the following literaturereferences: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2ndEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley& Sons, New York, 1987 and periodic updates; and the series Methods inEnzymology, Academic Press, San Diego.

A nucleic acid according to the present invention may include anypolymer or oligomer of pyrimidine and purine bases, preferably cytosine,thymine, and uracil, and adenine and guanine, respectively (See AlbertL. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)which is herein incorporated by reference in its entirety for allpurposes). The present invention contemplates any deoxyribonucleotide,ribonucleotide or peptide nucleic acid component, and any chemicalvariants thereof, such as methylated, hydroxymethylated or glycosylatedforms of these bases, and the like. The polymers or oligomers may beheterogenous or homogenous in composition, and may be isolated fromnaturally occurring sources or may be artificially or syntheticallyproduced. In addition, the nucleic acids may be DNA or RNA, or a mixturethereof, and may exist permanently or transitionally in single-strandedor double-stranded form, including homoduplex, heteroduplex, and hybridstates.

(Synthetic) oligonucleotide: single-stranded DNA molecules havingpreferably from about 5 to about 150 bases, which can be synthesizedchemically are referred to as synthetic oligonucleotides. In general,these synthetic DNA molecules are designed to have a unique or desirednucleotide sequence, although it is possible to synthesize families ofmolecules having related sequences and which have different nucleotidecompositions at specific positions within the nucleotide sequence. Theterm synthetic oligonucleotide will be used to refer to DNA moleculeshaving a designed or desired nucleotide sequence.

“Targeted Nucleotide Exchange” or “TNE”. Targeted nucleotide exchange(TNE) is a process by which at least one synthetic oligonucleotide, atleast partially complementary to a site in a chromosomal or an episomalgene directs the reversal of a nucleotide at a specific site. TNE hasbeen described using a wide variety of oligonucleotides and targets.Some of the reported oligonucleotides are RNA/DNA chimeras, containterminal modifications to impart nuclease resistance.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention pertains to a method for targetedalteration of a duplex acceptor DNA sequence comprising a first DNAsequence and a second DNA sequence which is the complement of the firstDNA sequence.

The method comprises combining a/the duplex acceptor DNA sequence withat least two donor oligonucleotides, being a first oligonucleotide and asecond oligonucleotide. The first oligonucleotide comprises at least onedomain that is capable of hybridizing to the first DNA sequence andfurther comprises at least one mismatch with respect to the first DNAsequence. This at least one mismatch is positioned at most 2 nucleotidesfrom the 3′ end of said first oligonucleotide. Preferably the mismatchis positioned at most 1 nucleotide from the 3′ end of saidoligonucleotide, even more preferably the mismatch is 0 nucleotides fromthe 3′ end of said oligonucleotide, in other words, is at the 3′ end ofsaid oligonucleotide. The second oligonucleotide comprises at least onedomain that is capable of hybridizing to the second DNA sequence andfurther comprises at least one mismatch with respect to the second DNAsequence. This at least one mismatch is positioned at most 2 nucleotidesfrom the 3′ end of said second oligonucleotide. Preferably the mismatchis positioned at most 1 nucleotide from the 3′ end of saidoligonucleotide, even more preferably the mismatch is 0 nucleotides fromthe 3′ end of said oligonucleotide, in other words, is at the 3′ end ofsaid oligonucleotide. The at least one mismatch in the firstoligonucleotide is relative to a nucleotide in the first DNA sequence ofthe duplex acceptor DNA sequence and the at least one mismatch in thesecond oligonucleotide is relative to a nucleotide in the second DNAsequence of the duplex acceptor DNA, wherein said nucleotides occupycomplementary positions in the duplex acceptor DNA (i.e. can form a basepair in the duplex acceptor DNA). In other words, the at least twooligonucleotides target the same base pair in the duplex acceptor DNA,the mismatch of the first oligonucleotide targets the nucleotide in thefirst DNA sequence and the mismatch of the second oligonucleotidetargets the complementary nucleotide in the second DNA sequence in theduplex DNA.

In other words, there is provided a method for targeted alteration of aduplex acceptor DNA sequence comprising a first DNA sequence and asecond DNA sequence which is the complement of the first DNA sequence,the method comprising combining the duplex acceptor DNA with at leasttwo oligonucleotides wherein the first oligonucleotide comprises adomain that is capable of hybridizing to the first DNA sequence andfurther comprises a mismatch relative to a nucleotide in the first DNAsequence and the second oligonucleotide comprises a domain that iscapable of hybridizing to the second DNA sequence and further comprisesa mismatch relative to a nucleotide in the second DNA sequence andwherein said nucleotides in the first DNA sequence and the second DNAsequence occupy complementary positions in the duplex acceptor DNA (e.g.form a base pair in the duplex acceptor DNA), and wherein,independently, the mismatch in the first oligonucleotide and themismatch in the second oligonucleotide is positioned at most 2nucleotides from the 3′ end of said oligonucleotide.

As mentioned above, each oligonucleotide of the at least twooligonucleotides according to the invention comprises a domain that iscapable of hybridizing either to the first or the second DNA sequence(under conditions that allow hybridization, as known to the skilledperson). Preferably, the domain that is capable of hybridizing to thefirst DNA sequence comprises at least one mismatch with respect to thefirst DNA sequence, or the mismatch is positioned directly adjacent tosaid domain (as long as the mismatch is at most 2 nucleotides from the3′ end of said oligonucleotide). Preferably, the domain that is capableof hybridizing to the second DNA sequence comprises at least onemismatch with respect to the second DNA sequence, or the mismatch ispositioned directly adjacent to said domain (as long as the mismatch isat most 2 nucleotides from the 3′ end of said oligonucleotide).

The method according to the invention allows for the specific andselective alteration of one or more nucleotides at (a) specific site(s)of an acceptor DNA sequence by means of oligonucleotides directed toboth strands of the duplex DNA and each directed to a differentnucleotide of the same base-pair as in present in the duplex DNA.

In particular the targeted alteration can be performed within a targetcell containing the duplex acceptor DNA sequence by the introductioninto that cell of the at least two oligonucleotides according to theinvention, i.e. a first oligonucleotide having, in comparison to thefirst DNA sequence to which it may hybridize, at least one mismatch andwherein said at least one mismatch is positioned at most 2, preferablyat most 1 nucleotide from the 3′ end of said oligonucleotide and asecond oligonucleotide having, in comparison to the second DNA sequenceto which it may hybridize, at least one mismatch and wherein said atleast one mismatch is positioned at most 2, preferably at most 1nucleotide from the 3′ end of said oligonucleotide, and wherein themismatch in the first oligonucleotide and the mismatch in the secondoligonucleotide are each directed to a different nucleotide of the samebase-pair in the duplex DNA.

Most preferably said at least one mismatch is at the 3′ end of theoligonucleotide, even more preferably said at least one mismatch is atthe 3′ end in both oligonucleotides. The result of the method is thetargeted alteration in a strand of one or more nucleotides so that thesequence of the target DNA sequence is altered. The invention maypreferably be performed in vivo but may also be performed ex vivo or invitro.

Within the context of the current invention, the duplex DNA sequencecomprises a first DNA sequence and a second DNA sequence. The second DNAsequence is the complement of the first DNA sequence and pairs to it toform the duplex. For example, a complement of a first DNA sequence ATTT(in the 5′ to 3′ direction) is TAAA (in the 3′ to 5′ direction). Thissecond DNA sequence pairs with the first DNA sequence to form a duplex.In case the duplex DNA sequence is, for example, part of a gene, thefirst DNA sequence may be either on the sense strand or anti-sensestrand.

The DNA of the duplex DNA sequence may be any type of DNA, such asgenomic DNA, DNA derived from genomic DNA, linear DNA, artificialchromosomes, nuclear chromosomal DNA, organellar DNA, BACs, YACs,plasmid DNA, or episomal DNA. The DNA sequence may be part of an intronor an exon, coding or non-coding, regulating expression or not.

The oligonucleotides used in the method disclosed herein are preferablysingle stranded and comprise at least one domain that is capable ofhybridizing to either the first DNA sequence (the first oligonucleotide)or the second DNA sequence (the second oligonucleotide).

For each of the two oligonucleotide, and independently from each other,the at least one mismatch with respect to the DNA sequence to be alteredand which mismatch is positioned 0, 1 or 2 nucleotides from the 3′ endof the oligonucleotide, is either comprised in the domain that iscapable of hybridizing to the first (for the first oligonucleotide) orsecond (for the second oligonucleotide) DNA sequence or is directlyadjacent to the domain.

The at least one domain in the oligonucleotide may thus comprise atleast one mismatch with respect to the DNA sequence to be altered or isdirectly next/adjacent to the mismatch. In other words, theoligonucleotide comprises a domain consisting of adjacent nucleotidesthan can hybridize, under the conditions of the experiment, with thefirst or second DNA sequence of the duplex acceptor DNA sequence, andeither comprises a mismatch with respect to said first or second DNAsequence or the mismatch is positioned directly next to said domain (andwherein the mismatch is positioned 0, 1 or 2 nucleotides from the 3′ endof the oligonucleotide).

For example, if the domain is (in the 5′ to 3′ direction) positioned upto 3 nucleotides from the 3′ end, the mismatch may be directly next tothe domain 2 nucleotides from the 3′ end of the oligonucleotide. Forexample if the domain is (in the 5′ to 3′ direction) positioned up to 1nucleotide from the 3′ end, the mismatch can be comprised in the domain,e.g. localized 2 nucleotides from the 3′ end, of be directly adjacent tothe domain, i.e. localized 0 nucleotides from the 3′ end, in other wordsat the 3′ end of the oligonucleotide.

It is to be understood that choices with respect to the position of themismatch in each of the at least two oligonucleotides can be madeindependently from the other oligonucleotide. In other words, in casethe mismatch in the first oligonucleotide is, for example, at the 3′ endof said oligonucleotide, the mismatch in the second oligonucleotide notnecessarily has to be positioned at the 3′ end of said oligonucleotide,but may also be positioned, for example at most 2 nucleotides from the3′ end of said oligonucleotide.

It is to be understood by the skilled person that within the context ofthe current invention, and where reference is made to the mismatch orthe mismatch comprised in the domain that is capable of hybridizing withthe first or second DNA sequence, these include any mismatch comprisedin the domain or positioned directly adjacent to the domain, as long asthe mismatch is positioned 2, 1 or 0 nucleotides from the 3′ end of theoligonucleotide.

In preferred embodiments the first oligonucleotide comprises preferablyno more than one mismatch with respect to the first DNA sequence, and/orthe second oligonucleotide comprises preferably no more than onemismatch with respect to the second DNA sequence (both directed to adifferent nucleotide of a base-pair as present in the duplex DNA).

In certain embodiments, more than one mutation can be introduced intothe target DNA, either simultaneously or successively. Theoligonucleotide can accommodate more than one mismatch on eitheradjacent or on removed locations on the oligonucleotide. In certainembodiments the oligonucleotide can comprise two, three, four or moremismatch nucleotides which may be remote (i.e. non-adjacent). Theoligonucleotide can comprise further domains to accommodate this. Themismatches may be in the same or in different domains.

It will be understood by the skilled person that the oligonucleotidesaccording to the invention may further comprise non-hybridizing parts,in other words adjacent nucleotides that do not hybridize with the firstor second DNA sequence, for example as these parts are not complementaryto any sequence in the first or second DNA sequence.

In a preferred embodiment the first oligonucleotide comprises one domainthat is capable of hybridizing to the first DNA sequence and comprises,or is directly adjacent to at least one mismatch, preferable onemismatch, with respect to the DNA sequence to be altered and the secondoligonucleotide comprises one domain that is capable of hybridizing tothe second DNA sequence and comprises, or is directly adjacent to atleast one mismatch, preferable one mismatch, with respect to the DNAsequence to be altered, wherein the at least one mismatch in the firstoligonucleotide is relative to a nucleotide in the first DNA sequence ofthe duplex acceptor DNA sequence and wherein the at least one mismatchin the second oligonucleotide is relative to a nucleotide in the secondDNA sequence of the duplex acceptor DNA, and wherein said nucleotidesoccupy complementary positions in the duplex acceptor DNA (e.g. form abase pair in the duplex acceptor DNA).

In such embodiment the oligonucleotide may, in principal, comprise morethan one domain that is capable of hybridizing to the respective firstor second DNA sequence, however only one of the domains may comprise, orbe directly adjacent to, the at least one mismatch (or the onemismatch), as disclosed herein. In another preferred embodiment theoligonucleotide, preferably both oligonucleotides, comprise(s) only onedomain that can hybridize to the duplex DNA. Such domain is located nearor at the 3′ end of the oligonucleotide and includes the mismatch, or isdirectly adjacent to the mismatch.

The oligonucleotides that are used as donors in the method disclosedherein can vary in length but generally vary in length between 10 and500 nucleotides, with a preference for 11 to 100 nucleotides, preferablyfrom 15 to 90, more preferably from 20 to 70.

The domain may consist of at least 5 nucleotides, including themismatch, but may also consist of all nucleotides, including themismatch, of the oligonucleotide. In case the mismatch is directlyadjacent to the domain, the domain may consist of at least 5nucleotides, but may also consist of all nucleotides of theoligonucleotide, except for the mismatch. Domain(s) in theoligonucleotide are typically in the order of at least 5, 10, preferably15, 20, 25 or 30 nucleotides.

The oligonucleotides according to the invention comprise at least onemismatch that is positioned at most 2, preferably at most 1 nucleotidefrom the 3′ end of said oligonucleotide. Preferably said (at least one)mismatch is at the 3′ end of the oligonucleotide, most preferably said(at least one) mismatch is at the 3′ end of the oligonucleotide in boththe first oligonucleotide and the second oligonucleotide. A personskilled in the art understands what the term 3′ end encompasses. Asingle-stranded non-circular DNA molecule has two ends, the 3′ end andthe 5′ end (also referred to as “three prime end” and “five prime end”).

The 5′ end of a single strand nucleic acid designates that specificnucleotide of which the C-5 carbon atom forms the terminal carbon atomof the sugar-phosphate backbone. The C-5 carbon atom may or may not belinked to a phosphate group by a phosphodiester bond, but this phosphategroup in turn does not form any linkage with another nucleotide. The 3′end of a single strand nucleic acid designates that specific nucleotideof which the C-3 carbon atom, is not linked to any other nucleotides,whether by means of a phosphate diester bond or otherwise. The C-5 atomis the 5^(th) carbon atom of the ribose or deoxyribose molecule and doesnot form part of the furanose ring, starting counting from the C atomdirectly adjacent to both the oxygen of the furanose ring and thenucleobase. The C-3 atom is the 3^(rd) carbon atom of the ribose ordeoxyribose molecule and forms part of the furanose ring, startingcounting from 1 which is the C atom directly adjacent to both the oxygenof the furanose ring and the nucleobase.

The term “mismatch positioned 2 nucleotides from the 3′ end” indicatesthat the mismatch is two nucleotides from the nucleotide at the 3′terminus of the oligonucleotide. The term “mismatch positioned 1nucleotide from the 3′ end” indicates that the mismatch is onenucleotide from the nucleotide at the 3′ terminus of theoligonucleotide. The term “mismatch positioned 0 nucleotides from the 3′end” indicates that the mismatch is the nucleotide at the 3′ terminus ofthe oligonucleotide.

In a preferred embodiment of the method described herein, the mismatchin the first oligonucleotide or the mismatch in the secondoligonucleotide is, independently, positioned at most 1 nucleotide fromthe 3′ end of said oligonucleotide, more preferably said at least onemismatch is at the 3′ end of the oligonucleotide, preferably themismatch in both oligonucleotides is at the 3′ end of the respectiveoligonucleotides.

Also preferred in the method described herein is that the domain in thefirst oligonucleotide and/or in the second oligonucleotide comprises oris directly adjacent to the at least one mismatch.

In addition, preferably in the method described herein, the firstoligonucleotide is complementary to the first DNA sequence except forthe mismatch and/or the second oligonucleotide is complementary to thesecond DNA sequence except for the mismatch. In such embodiment thefirst oligonucleotide thus comprises one mismatch with respect to thefirst DNA sequence and the second oligonucleotide comprises one mismatchwith respect to the second DNA sequence (each direct to a differentnucleotide of a base-pair in the duplex DNA). Such oligonucleotide iscomplementary to the first or second DNA sequence over the entire lengthof the oligonucleotide except for the one mismatch positioned at most 2,preferably at most 1 nucleotide from the 3′ end of said oligonucleotide,most preferably said mismatch is at the 3′ end of the oligonucleotide.In another embodiment, the oligonucleotide is (in the 5′ to 3′direction) complementary to the first or second DNA sequence over theentire length of the oligonucleotide up to the position of the mismatch(localized 2, 1 or 0 nucleotides from the 3′ end). Even more preferably,the mismatch in the first oligonucleotide is at the 3′ end and themismatch in the second oligonucleotide is at the 3′ end, and the firstoligonucleotide is complementary to the first DNA sequence over theentire length of the oligonucleotide, except for the mismatch, and thesecond oligonucleotide is complementary to the second DNA sequence overthe entire length of the oligonucleotide, except for the mismatch.

In another preferred embodiment of the method described herein, thefirst oligonucleotide and/or the second oligonucleotide comprises atleast one section that contains at least one modified nucleotide,wherein the modification is selected from the group consisting of a basemodification, a 3′ and/or 5′ end base modification, a backbonemodification or a sugar modification.

The base modification, 3′ and/or 5′ end base modifications, backbonemodification, and/or sugar modifications can be incorporated into theoligonucleotides to increase the (binding/hybridization) affinity of theoligonucleotides to the target sequence and, either independently oradditionally, to increase the oligonucleotides resistance againstcellular nucleases. However, as shown in example 2, it is not requiredthat the first or the second oligonucleotide incorporates any modifiednucleotide.

Any modification of a nucleotide in an oligonucleotide that provides anoligonucleotide suitable for use in the method according to theinvention (and comprising at least one mismatch positioned at most 2,preferably at most 1 nucleotide from the 3′ end of said oligonucleotide,most preferably said mismatch is at the 3′ end of the oligonucleotide)can advantageously be used. It will be understood by the skilled personthat a modification is relative to any one of a naturally occurring A,C, T, G nucleotides.

Advantageously, although not essential to the invention, the firstand/or the second oligonucleotide for use in the method according to theinvention may comprise modified nucleotides. In case both the first andthe second oligonucleotide comprise modification(s), the modificationsof the first may be the same as or different from the modifications ofthe second. In particular, any of the modifications discussed below maybe incorporated in the first and/or the second oligonucleotide accordingto the invention.

For example, the first and/or the second oligonucleotide may comprisemodification(s) that increase the resistance of the oligonucleotideagainst cellular nucleases, if compared to naturally occurring A, T, C,and G nucleotides. These modifications may include base modifications,backbone modifications, and/or sugar modifications. Typically, suchmodified nucleotides that increase the resistance of the oligonucleotideagainst cellular nucleases may result in an increased stability of theoligonucleotide in a cellular environment, which may result in improvedtargeted nucleotide exchange. Preferably, the first and/or the secondoligonucleotides for use according to the method of the inventioncomprises at least 1, preferably at least 2, more preferably at least 4,more preferably at least 6, most preferably at least 8 modifiednucleotides that increase the resistance of the oligonucleotide againstcellular nucleases if compared to naturally occurring A, T, C, and Gnucleotides. Alternatively, or at the same time, the first and/or thesecond oligonucleotide for use according to the method of the inventioncomprises at most 25, preferably at most 20, more preferably at most 15,most preferably at most 10 modified nucleotides that increase theresistance of the oligonucleotide against cellular nucleases if comparedto naturally occurring A, T, C, and G nucleotides. Such modifiednucleotides may be positioned at any position within the first and/orthe second oligonucleotide, preferably within 20 nucleotides, preferablywithin 15, more preferably within 10, even more preferably within 8,even more preferably within 6 nucleotides from the 3′ end and/or 5′ endof the respective oligonucleotide, and most preferably at the lastnucleotides at the 3′ end and/or at the last nucleotides at the 5′ end.As the mismatch which is to be incorporated in the target DNA sequenceis located zero, one, or at most two nucleotide(s) from the 3′ end ofthe oligonucleotides, it is particularly preferred that such modifiednucleotide(s) protect the 3′ side against cellular nucleases and thusare positioned on the 3′ end of the first and/or the secondoligonucleotide, such as within 20, 15, 10, 9, 8, 7, 6, or 4 nucleotidesfrom the 3′ end. However, as described earlier, and as shown in example2, it is not essential to the invention that the oligonucleotide indeedincludes modified nucleotides that increase resistance of theoligonucleotide against cellular nucleases.

Various of such modified nucleotides are mentioned herein, whichincrease the resistance of the oligonucleotide against cellularnucleases if compared to naturally occurring A, T, C, and G nucleotidesand which may be incorporated in the first and/or the secondoligonucleotide for use in the method according to the invention. Suchmodified nucleotide may be a nucleotide having phosphorothioatelinkage(s), but may also be a phosphoramidite, a methylphosphonate, or anucleotide with nonphosphate internucleotide bonds such as carbonates,carbamates, siloxane, sulfonamides and polyamide nucleic acid. Also, themodified nucleotides conferring cellular nuclease resistance asdescribed in WO0226967 may be used, such as LNA (Locked Nucleic Acid),or any other modified nucleotide that improves cellular nucleaseresistance of the oligonucleotide as known by the skilled person.

Alternatively or additionally to the above-described nuclease resistanceconferring modified nucleotides, the first and/or the secondoligonucleotide for use in the method according to the invention maycomprise modified nucleotides having a higher binding affinity to thetarget DNA sequence if compared to naturally occurring A, T, C, and Gnucleotides. These modification may include base modifications, backbonemodifications, and/or sugar modifications. Typically, such modifiednucleotides having increased binding affinity will affect strongerbase-pairing with the target sequence, which may result in an increasedstability of the hybrid between the oligonucleotide and the targetsequence, which is believed to result in improved targeted nucleotideexchange. Preferably, the first and/or the second oligonucleotide foruse according to the method of the invention comprises at least 1-10,preferably 1-8, more preferably 1-6, even more preferably 1-4, such as1, 2, 3, or 4, even more preferably 2 modified nucleotides having ahigher binding affinity to the target DNA sequence if compared tonaturally occurring A, T, C, and G nucleotides. Such modifiednucleotides as mentioned above may be positioned at any position withinthe first and/or the second oligonucleotide, preferably at a positionone nucleotide away from the mismatch, preferably at most 2, 3, 4, 5, 6,or 7 nucleotides away from the mismatch. Preferably, such modifiednucleotide is located at the 5′ side of the mismatch, but it may also beopted to position such modified nucleotide at the 3′ side of themismatch if the mismatch is not positioned at the last nucleotide at the3′ end of the first and/or the second oligonucleotide.

Various examples of such modified nucleotides having a higher bindingaffinity to the target DNA sequence if compared to naturally occurringA, T, C, and G nucleotides are mentioned herein which may beincorporated in the oligonucleotide for use in the method according tothe invention, including 2-OMe substitution, LNA (Locked Nucleic Acid),ribonucleotide, superA, superT, or any other type of modified nucleotidethat improves binding affinity of the oligonucleotide to the target DNAsequence if compared to naturally occurring A, T, C, and G nucleotides,as known by the skilled person.

Determining whether a modified nucleotide confers increased resistanceagainst cellular nucleases if compared to naturally occurring A, T, G, Cnucleotides may for example be done by comparing half-life times of aoligonucleotide having said modified nucleotide with a oligonucleotidenot having said modified nucleotide, in the presence of cellularnucleases as e.g. present in tomato extract, tomato cells, or in E.coli. If the half-life time of the first mentioned is higher, saidmodified nucleotide confers increased resistance against cellularnucleases if compared to naturally occurring A, T, G, C nucleotides.Determining whether a modified nucleotide confers higher bindingaffinity to the target DNA sequence if compared to naturally occurringA, T, C, or G nucleotides may for example be done by comparing meltingtemperature (Tm) of the duplex formed between the oligonucleotide havingsaid modified nucleotide and its target over that formed by theoligonucleotide not having said modified nucleotide and its target. Ifthe melting temperature of the first mentioned is higher, said modifiednucleotide confers higher binding affinity to the target DNA sequence ifcompared to naturally occurring A, T, G, C nucleotides.

A section according to the present invention is to be understood to beany part of the oligonucleotide with a length of at least onenucleotide. For example, a section may comprise 1-10, preferably 1-6,more preferably 1-4, more preferably 1-2 nucleotides, and may bepositioned at the 3′ side and/or the 5′ side of the mismatch. The atleast one section can be part of a domain according to the invention; inother words the section may be in a domain that can hybridize with thefirst or second DNA sequence. Alternatively, the section may overlapwith a domain, either completely or partially. In case of completeoverlap the section may have the same length of the domain, but may alsohave a length with exceeds the length of the domain. In the case ofpartial overlap, the domain and the section share at least onenucleotide.

Depending on the type of modification used in the oligonucleotide theremay be a preference for the modified nucleotide to be part of a domainthat can hybridize with the first or second DNA sequence, and whichdomain comprises or is directly adjacent to the at least one mismatchpositioned at most 2, preferably at most 1 nucleotide from the 3′ end ofsaid oligonucleotide, most preferably said mismatch is at the 3′ end ofthe oligonucleotide. This is in particular the case for modifiednucleotides with a higher binding affinity compared to naturallyoccurring A, C, T or G nucleotides with its complementary nucleotide.

Base modifications include, but are not limited to such modifications asfor example described in WO0226967, including modifications at the C-5position of pyrimidines such as 2′-deoxyuridine,5-fluoro-2′-deoxyuridine, 5-bromo-2′-deoxyuridine and5-methyl-2′-deoxycytidine. Other base modifications include syntheticand natural nucleobases like 5-methylcytosine, 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanineand 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanineand 3-deazaadenine.

End (3′ and/or 5′) modifications may include 2′-O-methyl bases, 3′ aminegroups, phosphorothioate linkages, or any other modification that isnuclease resistant. The skilled person is well aware of these kinds ofmodifications. Providing resistance to nuclease is believed to furtherimprove the targeted nucleotide exchange.

Various backbone modifications, such as those mentioned in WO0226967,including phosphorothioates, phosphoramidites and methylphosphonates,and those with nonphosphate internucleotide bonds, such as carbonates,carbamates, siloxane, sulfonamides and polyamide nucleic acid willincrease the resistance to cellular nucleases. Such backbonemodifications are therefore useful in the oligonucleotide used in themethod according to the invention.

In addition, sugar modifications, including but not limited to2′-O-methyl, 2′-fluoro or 2′-methoxyethoxy can increase thethermodynamic stability of a formed duplex, and at the same time provideimproved nuclease resistance.

Other examples of suitable modifications are described in WO2007073149.Modification of the donor oligonucleotides can for example comprisephosphorothioate linkages, 2-OMe substitutions, the use of LNAs (Lockednucleic acids), ribonucleotide and other bases that modify andpreferably enhance, the stability of the hybrid between theoligonucleotide and the acceptor stand either by improving affinitybinding to the target DNA or by inhibition of nuclease activity, orboth.

All these types of modifications are well know to the skilled person andare readily available from various commercial sources. It will beunderstood by the skilled person that modification can be introduced inthe first oligonucleotide independently of the second oligonucleotideused in the method described herein. For example, the firstoligonucleotide may comprise such modifications as described above,whereas the second oligonucleotide does not. Alternatively the firstoligonucleotide may comprise more, less or different modification at thesame or at different positions in the oligonucleotide in comparison tothe second oligonucleotide.

In an embodiment there is provided for a method according to theinvention wherein a modified nucleotide is incorporated in theoligonucleotide, or in both, and wherein the modified nucleotide has ahigher binding affinity compared to naturally occurring A, C, T, or Gnucleotides with its complementary nucleotide, and wherein the modifiednucleotide binds stronger to a nucleotide in the opposite position inthe first or second DNA sequence as compared to a naturally occurringnucleotide complementary to the nucleotide in the opposite position inthe first or second DNA sequence and/or wherein the modified nucleotideis a nuclease resistant nucleotide.

Preferably the modification is a base modification, a 3′ end and/or 5′end base modification, a backbone modification or a sugar modification.As discussed above, the donor oligonucleotides according to theinvention may contain modifications to improve the hybridizationcharacteristics such that the donor exhibits increased affinity for thetarget DNA strand, which may make intercalation of the donor easierand/or increases the thermodynamic stability of the formed duplex (incomparison to the same oligonucleotide not comprising such modification,and under the same experimental circumstances). The donoroligonucleotide can independently or in addition be modified to becomemore resistant against nucleases, which may stabilize the duplexstructure.

In the prior art a wide variety of modified nucleotides having a higherbinding affinity compared to naturally occurring A, C, T, or Gnucleotides with its complementary nucleotide, and wherein the modifiednucleotide binds stronger to a nucleotide in the opposite position inthe first or second DNA sequence as compared to a naturally occurringnucleotide complementary to the nucleotide in the opposite position inthe first or second DNA sequence and/or wherein the modified nucleotideis a nuclease resistant nucleotide have been described (see for ExampleWO 2007073154 and the various modifications discussed above).

In certain embodiments, a modification is at a position one nucleotideaway from to the mismatch, preferably 2, 3, 4, 5, 6 or 7 nucleotidesaway from the mismatch. In certain embodiments, modification is locatedat a position downstream from the mismatch. In certain embodiments,modification is located at a position upstream from the mismatch.

The domain that contains or is directly adjacent to the mismatch and thesections containing the modified nucleotide(s) may be overlapping. Thus,in certain embodiments, the domain containing the mismatch or directlyadjacent to the mismatch is located at a different position on theoligonucleotide than the section of which the modification isconsidered. In certain embodiments, the domain incorporates one or moresections. In certain embodiments, sections can incorporate the domain.In certain embodiments, the domain and the sections may be located atthe same position on the oligonucleotide and have the same length i.e.the sections coincide in length and position. In certain embodiments,there can be more than one section within a domain.

For the present invention, this means that the part of theoligonucleotide that contains the mismatch which is to alter the DNAduplex can be located at a different or shifted position from the partof the oligonucleotide that is modified.

Again, it will be understood by the skilled person that modificationscan be introduced in the first oligonucleotide independently of thesecond oligonucleotide used in the method described herein. For example,the first oligonucleotide may comprise such modifications as describedabove, whereas the second oligonucleotide does not. Alternatively thefirst oligonucleotide may comprise more, less or different modificationsat the same or at different positions in the oligonucleotide incomparison to the second oligonucleotide.

In a preferred embodiment the modified nucleotide is selected from thegroup consisting of LNAs and/or nucleotides having phosphorothioatebonds/linkage.

In a preferred embodiment, the modified nucleotide is a Locked NucleicAcid. Locked Nucleic Acid (LNA) is a DNA analogue with interestingproperties for use in antisense gene therapy and is known to the skilledperson.

LNAs are bicyclic and tricyclic nucleoside and nucleotide analogues andmay be incorporated in oligonucleotides. The basic structural andfunctional characteristics of LNAs and related analogues are disclosedin various publications and patents, including WO99/14226, WO00/56748,WO00/66604, WO98/39352, U.S. Pat. No. 6,043,060, and U.S. Pat. No.6,268,490, all of which are incorporated herein by reference in theirentireties.

LNA nucleosides are available for all the common nucleobases (T, C, G,A, U; for example from Exiqon (www.exiqon.com)) and are able to formbase pairs according to standard Watson-Crick base pairing rules. Whenincorporated into a DNA oligonucleotide, LNA makes the pairing with acomplementary nucleotide strand more rapid and increases the stabilityof the resulting duplex. In other words, LNA combines the ability todiscriminate between correct and incorrect targets (high specificity)with very high bio-stability (low turnover) and unprecedented affinity(very high binding strength to target). In fact, the affinity increaserecorded with LNA leaves the affinities of all previously reportedanalogues in the low-to-modest range.

LNA is an RNA analogue, in which the ribose is structurally constrainedby a methylene bridge between the 2′-oxygen and the 4′-carbon atoms.This bridge restricts the flexibility of the ribofuranose ring and locksthe structure into a rigid bicyclic formation. This so-called N-type (or3′-endo) conformation results in an increase in the Tm (meltingtemperature) of LNA containing duplexes, and consequently higher bindingaffinities and higher specificities. Importantly, the favorablecharacteristics of LNA do not come at the expense of other importantproperties as is often observed with nucleic acid analogues.

LNA can be mixed freely with all other chemistries that make up the DNAanalogue universe. LNA bases can be incorporated into oligonucleotidesas short all-LNA sequences or as longer LNA/DNA chimeras. LNAs can beplaced in internal, 3′ or 5′-positions. However, due to their rigidbicyclic conformations, LNA residues sometimes disturb the helical twistof nucleic acid strands. It is hence generally less preferred to designan oligonucleotide with two or more adjacent LNA residues. Preferably,the LNA residues are separated by at least one (modified) nucleotidethat does not disturb the helical twist, such as a conventionalnucleotide (A, C, T, or G).

The originally developed and preferred LNA monomer (the [beta]-D-oxy-LNAmonomer) has been modified into new LNA monomers. The novel[alpha]-L-oxy-LNA has been suggested to show superior stability against3′ exonuclease activity, and is also more powerful and more versatilethan [beta]-D-oxy-LNA in designing potent antisense oligonucleotides.Also xylo-LNAs, L-ribo LNAs and other LNA's can be used, as disclosed inWO9914226, WO00/56748, WO00/66604 and J. Org. Chem., 2010, 75 (7), pp2341-2349. In the present invention, any LNA of the above types iseffective in achieving the goals of the invention, i.e. improvedefficiency of TNE, with a preference for [beta]-D-LNA analogues.

As mentioned above, preferably, an LNA is at least one nucleotide awayfrom a mismatch in a (or both of the at least two oligonucleotides)oligonucleotide used in the method according to the invention. Althoughin the art on TNE, LNA modification has been listed amongst a list ofpossible oligonucleotide modifications as alternatives for the chimericmolecules used in TNE, it has been found that when single-stranded DNAoligonucleotides, as used in the method according to the invention, aremodified to contain LNA, TNE efficiency increase significantly to theextent that has presently been found when the LNA is positioned at leastone nucleotide away from the mismatch, even more preferably onenucleotide from the mismatch. The oligonucleotide preferably does notcontain more than about 75% (rounded to the nearest whole number ofnucleotides) LNAs.

In another preferred embodiment, the modified nucleotide comprises anucleotide having a phosphorothioate linkage. Many of the nucleotidemodifications commercially available have been developed for use inantisense applications for gene therapy. The simplest and most widelyused nuclease-resistant chemistry available for antisense applications(the “first generation” antisense-oligonucleotide) is thephosphorothioate (PS) linkage. In these molecules, a sulfur atomreplaces a non-bridging oxygen in the oligonucleotide phosphate backbone(see, for example, FIG. 2 of WO2007073154, resulting in resistance toendonuclease and exonuclease activity.

For gene therapy, a phosphorothioate/phosphodiester chimera generallyhas one to four PS-modified internucleoside linkages on both the 5′- and3′-ends with a central core of unmodified DNA. The phosphorothioatebonds can be incorporated, however, at any desired location in theoligonucleotide.

Preferably the modified nucleotide is an LNA or, even more preferably anucleotide having a phosphorothioate linkage, more preferably themodified oligonucleotide having at least one, for example, one, two,three or four, phosphorothioate(s). Preferably the oligonucleotidecontains at least one phosphorothioate at or near (e.g. within1,2,3,4,5,6,7 nucleotides from) the 5′ end of the oligonucleotideaccording to the invention.

In an embodiment there is provided that the oligonucleotide used in themethod according to the invention comprises at least two, three, four,or five modified nucleotides. Preferably the oligonucleotide comprisestwo, three four or five modified nucleotides. Preferably themodifications are selected from the group consisting of LNAs and/orphosphorothioate bonds.

In certain preferred embodiments of the invention, the nucleotide in theoligonucleotide at the position of the mismatch can be modified. Whetheror not the mismatch can be modified will depend to a large extent on theexact mechanism of the targeted nucleotide exchange or of the cell's DNArepair mechanism using the difference in affinity between the donor andacceptor strands. In a preferred embodiment the nucleotide at theposition of the mismatch is not a modified nucleotide.

In an embodiment there is provided for a method according to theinvention wherein the modified nucleotide is at least one nucleotidefrom the at least one mismatch located at most 2, preferably at most 1nucleotide from the 3′ end of said oligonucleotide, most preferably saidat least one mismatch is at the 3′ end of the oligonucleotide.

As discussed previously, it has been found that when single-stranded DNAoligonucleotides, as used in the method according to the invention, aremodified to contain modified nucleotides, for example LNA, TNEefficiency increases significantly to the extent that has presently beenfound when the modified nucleotide, preferably LNA, is positioned atleast one nucleotide away from the mismatch, even more preferably onemismatch from the mismatch. In other words, in a preferred embodiment, amodified nucleotide, preferably a LNA, is separated from the mismatch byat least one other nucleotide, which at least one other nucleotide isnot a LNA, preferably not a modified nucleotide. However, in case of forexample a phosphorothioate linkage, such linkage by be directly adjacentto the mismatch nucleotide.

In an embodiment there is provided for a method wherein the alterationof the duplex acceptor DNA is within a cell preferably selected from thegroup consisting of a prokaryotic cell, a bacterial cell, a eukaryoticcell, a plant cell, an animal cell, a yeast cell, a fungal cell, arodent cell, a human cell, a non-human cell, and/or a(n) (non-human)embryonic cell. The invention is, in its broadest form, genericallyapplicable to all sorts of organisms such as humans, animals, plants,fish, reptiles, insects, fungi, bacteria and so on. The invention canthus be performed within a cell selected from the group consisting of aprokaryotic cell, a bacterial cell, a eukaryotic cell, a plant cell, ananimal cell, a yeast cell, a fungal cell, a rodent cell, a human cell, anon-human cell, and/or an embryonic cell. In a preferred embodiment, thecell is a plant cell.

There is also provided for a method as described herein wherein theduplex acceptor DNA is obtained from a prokaryotic organism, bacteria, aeukaryotic organism, a plant, an animal, a yeast, a fungus, a rodent, ora human. In a preferred embodiment the duplex acceptor DNA is obtainedfrom a plant (or is plant DNA present in a plant cell).

In an embodiment of the invention, the alteration of the duplex acceptorDNA sequence is a deletion, a substitution and/or an insertion of atleast one nucleotide. Preferably the alteration of the duplex DNAsequence is a deletion, a substitution and/or an insertion of no morethan 5 nucleotides, preferably no more than 4, 3, 2, 1 nucleotide(s),most preferably one nucleotide (or in other words, one base-pair ismodified in the duplex DNA). More preferably the alteration of theduplex acceptor DNA sequence is a substitution of no more than 5nucleotides, preferably no more than 4, 3, 2, 1 nucleotide(s), mostpreferably one nucleotide.

In another embodiment there is provided a method according to theinvention, wherein the duplex acceptor DNA is from genomic DNA, linearDNA, artificial chromosomes, mammalian artificial chromosomes, bacterialartificial chromosomes, yeast artificial chromosomes, plant artificialchromosomes, nuclear chromosomal DNA, organellar DNA, and/or episomalDNA including plasmids.

Indeed the invention is applicable for the modification of any type ofDNA, such as those disclosed above. The invention can be performed invivo as well as ex vivo or in vitro, for example by subjecting the DNAto be modified with the donor oligonucleotide in the presence ofproteins that are capable of targeted nucleotide exchange, for instance,and in particular, proteins that are functional in the mismatch repairmechanism of the cell.

The delivery of the oligonucleotide to a cell can be achieved viaelectroporation or other conventional techniques that are capable ofdelivering either to the nucleus or the cytoplasm. In vitro testing ofthe method of the present invention can be achieved using the Cell Freesystem as is described i.a. in WO01/87914, WO03/027265, WO99/58702,WO01/92512. The oligonucleotide may comprise methylated nucleotides,non-methylated nucleotides or both.

The invention is, in its broadest form, applicable for many purposes foraltering a cell, correcting a mutation by restoration to wild type,inducing a mutation, inactivating an enzyme by disruption of codingregion, modifying bioactivity of an enzyme by altering coding region,modifying a protein by disrupting the coding region.

The invention also relates to the use of oligonucleotides essentially asdescribed hereinbefore, for altering a cell, correcting a mutation byrestoration to wild type, inducing a mutation, inactivating an enzyme bydisruption of coding region, modifying bioactivity of an enzyme byaltering coding region, modifying a protein by disrupting the codingregion, mismatch repair, targeted alteration of (plant) geneticmaterial, including gene mutation, targeted gene repair and geneknockout. Preferably the method according to the invention is fortargeted alteration of duplex acceptor DNA obtained from a plant,present in a plant, or to be presented to a plant.

The invention further relates to kits, preferably comprising at leastone, preferably both of the oligonucleotides used in the methodaccording to the invention, and as defined herein, optionally incombination with proteins that are capable of TNE.

In particular, the kit comprises instructions for targeted alteration ofa duplex DNA in accordance with the method described and claimed herein.The instructions comprise essentially a description of the steps of themethod according to the invention described herein.

In particular there is provided a kit comprising instructions forperforming a method for targeted alteration of a duplex acceptor DNAaccording to the invention and as disclosed herein, wherein the kitfurther comprises at least two oligonucleotides for use in the method asdescribed herein, preferably the at least two oligonucleotides asdescribed herein.

In this embodiment, the kit may thus comprise at least a first and asecond oligonucleotide that, independently, each comprise at least onedomain that is capable of hybridizing to, respectively, the first orsecond DNA sequence and which domain comprises, or is directly adjacentto at least one mismatch with respect to, respectively, the first orsecond DNA sequence, and wherein said at least one mismatch ispositioned at most 2, preferably at most 1 nucleotide from the 3′ end ofsaid oligonucleotide, most preferably said at least one mismatch is atthe 3′ end of the oligonucleotide, and wherein the mismatch in the firstoligonucleotide and the mismatch in the second oligonucleotide eachtarget a different nucleotide, wherein the nucleotide targeted in thefirst strand occupies the complementary position of the targetednucleotide in the second strand, e.g. the nucleotides form a base-pairin the duplex DNA, and in addition comprises instructions to perform themethod according to the invention.

As will be understood by the skilled person, by providing instructionsat least informing that the above mismatch is positioned at the 3′ endof the oligonucleotide(s) or 1 nucleotide from the 3′ end or 2nucleotides from the 3′ end, and that the oligonucleotide(s) can be usedfor alteration of a duplex DNA sequence, such kit comprising theseinstructions and the oligonucleotide(s) are a kit within the scope ofthe above described and claimed kits.

The kit may, for example, also take the form of a website or a documentproviding instructions or information to perform targeted alteration ofa duplex acceptor DNA according to the method of the invention, asdescribed and disclosed herein, and the (separate) provision or offeringof an oligonucleotide(s) suitable for use in the method according to theinvention, and as described and disclosed herein.

In a preferred embodiment there is provided for a kit according to theinvention, as described above, wherein the oligonucleotide is anoligonucleotide that, when combined with a duplex acceptor DNA sequencecontaining a first DNA sequence and a second DNA sequence which is thecomplement of the first DNA sequence, comprises a domain that is capableof hybridizing to the first DNA sequence, which domain comprises, or isdirectly adjacent to, at least one mismatch with respect to the firstDNA sequence, and wherein said at least one mismatch is located at most2, preferably at most 1 nucleotide from the 3′ end of saidoligonucleotide, most preferably said at least one mismatch is at the 3′end of the oligonucleotide.

In a preferred embodiment there is provided a kit wherein, when combinedwith a duplex acceptor DNA sequence containing a first DNA sequence anda second DNA sequence which is the complement of the first DNA sequence,the first oligonucleotide comprises at least one domain that is capableof hybridizing to the first DNA sequence and wherein the firstoligonucleotide further comprises at least one mismatch with respect tothe first DNA sequence and wherein the at least one mismatch ispositioned at most 2 nucleotides from the 3′ end of said firstoligonucleotide; and wherein the second oligonucleotide comprises atleast one domain that is capable of hybridizing to the second DNAsequence and wherein the second oligonucleotide further comprises atleast one mismatch with respect to the second DNA sequence and whereinthe at least one mismatch is positioned at most 2 nucleotides from the3′ end of said second oligonucleotide; and wherein the at least onemismatch in the first oligonucleotide is relative to a nucleotide in thefirst DNA sequence of the duplex acceptor DNA sequence and wherein theat least one mismatch in the second oligonucleotide is relative to anucleotide in the second DNA sequence of the duplex acceptor DNA, andwherein said nucleotides occupy complementary positions in the duplexacceptor DNA (for example, form a base pair in the duplex acceptor DNA).

As will be understood by the skilled person, in a preferred embodiment,the mismatch in the first oligonucleotide and the mismatch in the secondoligonucleotide, each directed to a different nucleotide in a (the same)base-pair in the duplex DNA, is preferably such that when bothmismatches would be introduced in the duplex DNA, these arecomplementary to each other and may form a base-pair (A-T/C-G) in theduplex DNA in which they are introduced.

Examples Example 1: TNE on a GFP Episome in Tobacco Protoplasts Using 2Oligonucleotides

TNE involves the introduction of oligonucleotides into cells where theyinduce a mutation in the genomic target locus, driven by a mismatchnucleotide in the oligonucleotide.

In the experiments below accuracy and efficiency of TNE was determinedby performing TNE on an episome (plasmid) which carries a non-functionalGreen fluorescent protein (GFP) containing an in frame stop codon. Twooligonucleotides were designed each carrying at the 3′ end a mismatchnucleotide which could repair the stop codon in GFP. Co-transfection ofthe plasmid together with the two oligonucleotides restored GFPexpression and activity which was, in the experiments below, scored at asingle cell level 24 hours after protoplast transfection. This firstexample describes experiments performed in tobacco protoplasts.

Materials and Methods Constructs

The functional GFP open reading frame was synthesized and the codonusage was optimized for use in the Solanaceae. A variant of GFP wasproduced with a nucleotide change at position 82 (G to T) as shown inFIG. 1. This resulted in the production of an in frame stop codon andthe amino acid sequence of the resulting protein is shown in FIG. 2. TheGFP ORF (GFP WT) and GFP variant with the stop codon (GFP-STOP) werecloned as XhoI-SacI fragments in the multiple cloning site of a pUCbased vector containing the CaMV 35S promoter for gene expression inplant cells. This resulted in the constructs pKG7381 (GFP-WT) andpKG7384 (GFP-STOP). In addition, GFP is translationally fused to a 6×HIStag and an NLS (sequence nuclear localization signal) to facilitateaccumulation of GFP protein in the protoplast nucleus and thus improveour ability to score GFP positive cells. These constructs are shown inFIG. 3.

Oligonucleotides

The oligonucleotides to repair the stop codon in the GFP gene are shownin Table 1.

TABLE 1 Oligonucleotides used in this study. Oligo Sequence OrientationODM1 G*T*T*C*TCGAGATGGTGAGCAAG*G*G*C*T Sense (SEQ ID NO: 3) ODM2G*C*A*C*CACCCCGGTGAACAGCT*C*C*T*A Antisense (SEQ ID NO: 4) ODM3G*T*T*C*TCGAGATGGTGAGCAAG*G*G*C*G Sense (SEQ ID NO: 5) ODM4G*C*A*C*CACCCCGGTGAACAGCT*C*C*T*C Antisense (SEQ ID NO: 6) The mismatchnucleotide in ODM3 and ODM4 is underlined. The asterisks representphosphorothioate (PS) linkages. The orientation of the oligonucleotideis given as sense (identical to the GFP coding sequence) or antisense(complementary to the GFP coding sequence). All oligonucleotides areshown in the 5′-3′ orientation.

Isolation and Transfection of Tobacco Protoplasts

The source material for this example was tobacco in vitro shootcultures, grown aseptically in glass jars (750 ml) in MS20 medium at atemperature of 25/20° C. (day/night) and a photon flux density of 80μE·m⁻²·s⁻¹ (photoperiod of 16/24 h). MS20 medium is basic Murashige andSkoog's medium (Murashige, T. and Skoog, F., Physiologia Plantarum, 15:473-497, 1962) containing 2% (w/v) sucrose, no added hormones and 0.8%Difco agar. The shoots were subcultured every 3 weeks to fresh medium.

For the isolation of mesophyll protoplasts, fully expanded leaves of 3-6week old shoot cultures were harvested. The leaves are sliced into 1 mmthin strips, which were then transferred to large (100 mm×100 mm) Petridishes containing 45 ml MDE basal medium for a preplasmolysis treatmentof 30 min at room temperature. MDE basal medium contained 0.25 g KCl,1.0 g MgSO₄.7H₂O, 0.136 g of KH₂PO₄, 2.5 g polyvinylpyrrolidone (MW10,000), 6 mg naphthalene acetic acid and 2 mg 6-benzylaminopurine in atotal volume of 900 ml. The osmolarity of the solution was adjusted to600 mOsm·kg⁻¹ with sorbitol, the pH to 5.7.

After preplasmolysis, 5 ml of enzyme stock was added to each Petri dish.The enzyme stock consisted of 750 mg Cellulase Onozuka R10, 500 mgdriselase and 250 mg macerozyme R10 per 100 ml (Duchefa B. V., Haarlem,The Netherlands, e.g. products C8001 & M8002), filtered over Whatmanpaper and filter-sterilized. The Petri dishes were sealed and incubatedovernight in the dark at 25° C. without movement to digest the cellwalls.

The protoplast suspension was then passed through 500 μm and 100 μmsieves into 250 ml Erlenmeyer flasks, mixed with an equal volume of KClwash medium, and centrifuged in 50 ml tubes at 85×g for 10 min. KCl washmedium consisted of 2.0 g CaCl₂.2H₂O per liter and a sufficient quantityof KCl to bring the osmolarity to 540 mOsm·kg⁻¹.

The centrifugation step was repeated twice, first with the protoplastsresuspended in MLm wash medium, which is the macro-nutrients of MSmedium (Murashige, T. and Skoog, F., Physiologia Plantarum, 15: 473-497,1962) at half the normal concentration, 2.2 g of CaCl₂.2H₂O per literand a quantity of mannitol to bring the osmolality to 540 mOsm·kg⁻¹, andfinally with the protoplasts resuspended in MLs medium, which is MLmmedium with mannitol replaced by sucrose.

The protoplasts were recovered from the floating band in sucrose mediumand resuspended in an equal volume of KCl wash medium. Their densitieswere counted using a haemocytometer. Subsequently, the protoplasts werecentrifuged again in 10 ml glass tubes at 85×g for 5 min and the pelletsresuspended at a density of 1×10⁵ protoplasts ml⁻¹ in electroporationmedium.

Protoplast Electroporation

A BioRad Gene Pulser apparatus was used for electroporation. Using PHBSas an electroporation medium (10 mM Hepes, pH 7.2; 0.2 M mannitol, 150mM NaCl; 5 mM CaCl2) and with a protoplast density in theelectroporation mixture of ca. 1×10⁶ per ml, the electroporationsettings were 250V (625 V cm⁻¹) charge and 800 μF capacitance with arecovery time between pulse and cultivation of 10 minutes. For eachelectroporation ca. 2 μg total oligonucleotide and 20 μg KG7381 orKG7384 were used per 800 microliter electroporation.

After the electroporation treatment, the protoplasts were placed on icefor 30 min to recover, then resuspended in T₀ culture medium at adensity of 1×10⁵ protoplasts ml⁻¹ and incubated at 21° C. overnight inthe dark. T₀ culture medium contained (per liter, pH 5.7) 950 mg KNO₃,825 mg NH₄NO₃, 220 mg CaCl₂.2H₂O, 185 mg MgSO₄.7H₂O, 85 mg KH₂PO₄, 27.85mg FeSO₄.7H₂O, 37.25 mg Na₂EDTA.2H₂O, the micro-nutrients according toHeller's medium (Heller, R., Ann Sci Nat Bot Biol Veg 14: 1-223, 1953),vitamins according to Morel and Wetmore's medium (Morel, G. and R. H.Wetmore, Amer. J. Bot. 38: 138-40, 1951), 2% (w/v) sucrose, 3 mgnaphthalene acetic acid, 1 mg 6-benzylaminopurine and a quantity ofmannitol to bring the osmolality to 540 mOsm·kg⁻¹ The protoplasts wereexamined under the UV microscope 20 hours after electroporation tovisualize the GFP signal in the nucleus.

Alternatively, PEG treatment could be used to introduce the plasmid andoligonucleotide DNA into tobacco protoplasts. Methods to achieve thisare well known in the literature.

Results

When the construct KG7381 (GFP-WT) was electroporated to tobaccoprotoplasts a strong GFP signal located in the nucleus afterapproximately 20 hours of incubation was observed. This signal is due tothe strong transient expression of the GFP ORF. This signal disappearedwithin 48 hours, presumably due to degradation/elimination of theplasmid DNA from the cell. In a typical experiment, approximately 30% ofthe protoplasts showed a GFP signal and this represents the maximalelectroporation efficiency. No GFP signal was observed when KG7384(GFP-STOP) was introduced into tobacco protoplasts.

Once the experimental setup had been validated, experiments wereperformed whereby KG7384 was introduced into tobacco protoplasts incombination with the oligonucleotides described above. The GFP signalwas scored after 24 hours and the results are shown in table 2.

TABLE 2 Repair of episomal GFP Treatment Oligonucleotide(s) Repairefficiency (%) 1 ODM1 0 2 ODM2 0 3 ODM3 20 4 ODM4 14 5 ODM1 + ODM2 0 6ODM3 + ODM4 80 Repair efficiency was calculated as the percentage ofcells with restored GFP expression scored via fluorescence.

When oligonucleotides lacking a mismatch at the 3′ end (ODM1 and ODM2)were added separately (treatment 1 & 2) or together (treatment 5) norestoration of GFP activity was observed. In contrast, we did observerestoration of GFP expression when oligonucleotides carrying a singlemismatch at the 3′ end (ODM3 and ODM4) were used (treatment 3 & 4).Surprisingly, we were able to demonstrate that the repair efficiency washigher than expected when ODM3 and ODM4 were added simultaneously.Therefore, such an approach appears to significantly improve theefficiency of TNE and enables the development of a more efficient TNEmethodology.

Example 2. Effects of PS Linkage on TNE Efficiency

This example shows that it is not required that the first or the secondoligonucleotide according to the invention incorporates nucleotideshaving e.g. phosphorothioate linkages nor that is it required that anyother type of modification is incorporated.

Methods

Tomato mesophyll protoplasts were isolated from young leaves of tomatoin vitro plants. Reporter constructs harbouring an eYFP(stop) gene (seeFIGS. 5 and 6) whose expression was driven by the CaMV 35S promoter andoligonucleotides were transfected into tomato protoplasts by aPEG-mediated method. After overnight incubation under dark at 30° C. ina growth chamber, infected protoplasts were observed using a fluorescentmicroscope equipped with a YFP filter set. The number of protoplastsemitting yellow fluorescence was scored and the TNE efficiency wascalculated by dividing the number of yellow protoplasts by the number oftransfected protoplasts.

Sequence of Oligonucleotides Tested:

(SEQ ID NO: 7) PB72 C*A*T*G*CATGCATGCATGCATGC*A*T*G*C25 mer, PS, Nonsense (= negative control) (SEQ ID NO: 8) PB242T*G*A*G*GGTGAAGGTGATGCTAC*T*T*A*C 25 mer, PS, 3′ MM (= mismatch) Sense(SEQ ID NO: 9) PB243 G*A*T*G*AACTTAAGTGTAAGTTT*A*C*C*G 25 mer, PS, 3′MM Antisense (SEQ ID NO: 10) TF7 TGAGGGTGAAGGTGATGCTACTTAC 25 mer, 3′MM Sense (SEQ ID NO: 11) TF8 GATGAACTTAAGTGTAAGTTTACCG 25 mer, 3′MM Antisense *represents a phosphorothioate linkage

The TNE reaction caused by PB242, PB243, TF7, and TF8 converts thetarget sequence from TAA to TAC. Oligonucleotides PB242, PB243, TF7 andTF8 were thus designed to repair the STOP codon in YFP, wherein PB72,PB242, and PB243 comprise PS linkages, and TF7 and TF8 do not comprisePS linkages. As shown in FIG. 4, PB242+PB243 was able to restore YFPexpression with more than 23%; TF7+TF8 was able to restore YFPexpression with more than 3%, almost 10 times more in comparison to thesignal obtained with the nonsense oligonucleotide.

This example thus shows that using oligonucleotides, with or withoutmodification, like PS linkages, can be used in TNE.

LITERATURE

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1. A method for targeted alteration of a duplex acceptor DNA sequencecomprising a first DNA sequence and a second DNA sequence which is thecomplement of the first DNA sequence, the method comprising combiningthe duplex acceptor DNA sequence with at least a first oligonucleotideand a second oligonucleotide, wherein the first oligonucleotidecomprises at least one domain that is capable of hybridizing to thefirst DNA sequence and wherein the first oligonucleotide furthercomprises at least one mismatch with respect to the first DNA sequenceand wherein the at least one mismatch is positioned at most 2nucleotides from the 3′ end of said first oligonucleotide; and whereinthe second oligonucleotide comprises at least one domain that is capableof hybridizing to the second DNA sequence and wherein the secondoligonucleotide further comprises at least one mismatch with respect tothe second DNA sequence and wherein the at least one mismatch ispositioned at most 2 nucleotides from the 3′ end of said secondoligonucleotide; and wherein the at least one mismatch in the firstoligonucleotide is relative to a nucleotide in the first DNA sequence ofthe duplex acceptor DNA sequence and wherein the at least one mismatchin the second oligonucleotide is relative to a nucleotide in the secondDNA sequence of the duplex acceptor DNA, and wherein said nucleotidesoccupy complementary positions in the duplex acceptor DNA.
 2. The methodaccording to claim 1 wherein the mismatch in the first oligonucleotideor the mismatch in the second oligonucleotide is, independently,positioned at most 1 nucleotide from the 3′ end of said oligonucleotide,more preferably said at least one mismatch is at the 3′ end of theoligonucleotide, preferably the mismatch in both oligonucleotides is atthe 3′ end of the oligonucleotides.
 3. The method according to claim 1wherein the domain in the first oligonucleotide and/or in the secondoligonucleotide comprises or is directly adjacent to the at least onemismatch.
 4. The method according to claim 1 wherein the firstoligonucleotide is complementary to the first DNA sequence except forthe mismatch and/or wherein the second oligonucleotide is complementaryto the second DNA sequence except for the mismatch.
 5. The methodaccording to claim 4 wherein the mismatch in the first oligonucleotideis at the 3′ end and wherein the mismatch in the second oligonucleotideis at the 3′ end.
 6. The method according to claim 1 wherein the firstoligonucleotide and/or the second oligonucleotide comprises at least onesection that contains at least one modified nucleotide, wherein themodification is selected from the group consisting of a basemodification, a 3′ and/or 5′ end base modification, a backbonemodification or a sugar modification.
 7. The method according to claim 6wherein the modified nucleotide is selected from the group consisting ofLNA or phosphorothioate bonds.
 8. The method according to claim 6wherein the oligonucleotide comprises at least two, three, four, or fivemodified nucleotides, preferably the oligonucleotide comprises two,three, four or five modified nucleotides.
 9. The method according toclaim 1 wherein the mismatch is not a modified nucleotide.
 10. Themethod according to claim 6 wherein the modified nucleotide is at leastone nucleotide from the at least one mismatch located at most 2,preferably at most 1 nucleotide from the 3′ end of said oligonucleotide,most preferably said at least one mismatch is at the 3′ end of theoligonucleotide.
 11. The method according to claim 1, wherein thealteration of the duplex acceptor DNA is within a cell preferablyselected from the group consisting of prokaryotic cell, a bacterialcell, a eukaryotic cell, a plant cell, an animal cell, a yeast cell, afungal cell, a rodent cell, a human cell, a non-human cell, and/or anembryonic cell.
 12. The method according to claim 1 wherein the duplexacceptor DNA is obtained from a prokaryotic organism, a bacteria, aneukaryotic organism, a plant, an animal, a yeast, a fungus, a rodent, ora human.
 13. The method according to claim 1, wherein the alteration isa deletion, a substitution and/or an insertion of at least onenucleotide.
 14. The method according to claim 1, wherein the duplexacceptor DNA is from genomic DNA, linear DNA, artificial chromosomes,mammalian artificial chromosomes, bacterial artificial chromosomes,yeast artificial chromosomes, plant artificial chromosomes, nuclearchromosomal DNA, organellar DNA, and/or episomal DNA including plasmids.15. The method according to claim 1, for altering a cell, correcting amutation by restoration to wild type, inducing a mutation, inactivatingan enzyme by disruption of coding region, modifying bioactivity of anenzyme by altering coding region, modifying a protein by disrupting thecoding region.
 16. (canceled)
 17. A kit comprising instructions forperforming a method for targeted alteration of a duplex acceptor DNAaccording to claim
 1. 18. A kit according to claim 17 further comprisingat least two oligonucleotides for use in the method according to claim1, preferably comprising the at least two oligonucleotides as describedin claim
 1. 19. A kit according to claim 17 wherein, when combined witha duplex acceptor DNA sequence containing a first DNA sequence and asecond DNA sequence which is the complement of the first DNA sequence,the first oligonucleotide comprises at least one domain that is capableof hybridizing to the first DNA sequence and wherein the firstoligonucleotide further comprises at least one mismatch with respect tothe first DNA sequence and wherein the at least one mismatch ispositioned at most 2 nucleotides from the 3′ end of said firstoligonucleotide; and wherein the second oligonucleotide comprises atleast one domain that is capable of hybridizing to the second DNAsequence and wherein the second oligonucleotide further comprises atleast one mismatch with respect to the second DNA sequence and whereinthe at least one mismatch is positioned at most 2 nucleotides from the3′ end of said second oligonucleotide; and wherein the at least onemismatch in the first oligonucleotide is relative to a nucleotide in thefirst DNA sequence of the duplex acceptor DNA sequence and wherein theat least one mismatch in the second oligonucleotide is relative to anucleotide in the second DNA sequence of the duplex acceptor DNA, andwherein said nucleotides occupy complementary positions in the duplexacceptor DNA.